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.

The nuclear lamina is part of the nuclear envelope (NE). Lamin filaments provide the nucleus with mechanical stability and are involved in many nuclear activities. The functional importance of these proteins is highlighted by mutations in lamin genes, which cause a variety of human diseases (laminopathies). Here we describe a method that allows one to quantify the contribution of lamin A protein to the mechanical properties of the NE. Lamin A is ectopically expressed in Xenopus oocytes, where it is incorporated into the NE of the oocyte nucleus, giving rise to a prominent lamina layer at the inner nuclear membrane. Nuclei are then isolated and probed by atomic force microscopy. From the resulting force curves, stiffness values are calculated and compared with those of control nuclei. Expression of lamin A significantly increases the stiffness of oocyte nuclei in a concentration-dependent manner. Since chromatin adds negligibly to nuclear mechanics in these giant nuclei, this method allows one to measure the contribution of individual NE components to nuclear mechanics.

We propose a magnetic force modulation method to measure the stiffness and viscosity of living cells using a modified AFM apparatus. An oscillating magnetic field makes a magnetic cantilever oscillate in contact with the sample, producing a small AC indentation. By comparing the amplitude of the free cantilever motion (A0) with the motion of the cantilever in contact with the sample (A1), we determine the sample stiffness and viscosity. To test the method, the frequency-dependent stiffness of 3T3 fibroblasts was determined as a power law k(s)(f) = α + β(f/f¯)(γ) (α = 7.6 × 10(-4) N m(-1), β = 1.0 × 10(-4) N m(-1), f¯ = 1 Hz, γ = 0.6), where the coefficient γ = 0.6 is in good agreement with rheological data of actin solutions with concentrations similar to those in cells. The method also allows estimation of the internal friction of the cells. In particular we found an average damping coefficient of 75.1 μN s m(-1) for indentation depths ranging between 1.0 μm and 2.0 μm.

We have measured the creep response of soft gels and cells after applying a step in loading force with atomic force microscopy (AFM). By analysing the creep response data using the standard linear solid model, we can quantify the viscous and elastic properties of these soft samples independently. Cells, in comparison with gels of similar softness, are much more viscous, as has been qualitatively observed in conventional force curve data before. Here, we quantify the spring constant and the viscous damping coefficient from the creep response data. We propose two different modes for applying a force step: (1) indirectly by increasing the sample height or (2) directly by employing magnetic cantilevers. Both lead to similar results, whereas the latter seems to be better defined since it resembles closely a constant strain mode. The former is easier to implement in most instruments, and thus may be preferable from a practical point of view. Creep analysis by step response is much more appropriate to analyse the viscoelastic response of soft samples like cells than the usually used force curve analysis.

Extended abstract of a paper presented at Microscopy and Microanalysis 2010 in Portland, Oregon, USA, August 1 – August 5, 2010.