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.

Measuring the viscoelastic creep of soft samples by step response AFM.

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.

Immobilisation and characterisation of the demineralised, fully hydrated organic matrix of nacre--an atomic force microscopy study.

Mimicry of the tough natural composite nacre in future bioengineering requires knowledge of the biomineralisation process. The insoluble organic matrix isolated from the shell of the gastropod Haliotis laevigata was characterised by protein chemistry, topographical and mechanical measurements. Demineralisation of nacre in dilute acetic acid or ethylenediaminetetraacetic acid revealed a set of soluble proteins and the insoluble matrix. The insoluble matrix contains a chitin core and firmly attached proteins, which could be removed by sodium dodecyl sulfate and glycerol indicating a hydrophobic interaction. Atomic force microscopy images of the native insoluble matrix showed a filamentous network with pores or holes, where the filaments showed globular attachments of different sizes, possibly the attached protein molecules. During direct observation of protein degradation imaged by atomic force microscopy the insoluble matrix gets smooth and flat indicating the removal of the attached proteins by proteases. We propose a model of protein coated chitin filaments for the insoluble matrix of nacre. Mechanical measurements by force mapping revealed a Young's modulus depending on the hydration state of the organic layers. The fully hydrated organic matrix has an elastic modulus below 1 MPa comparable to some hydrogels.

Keratocyte lamellipodial protrusion is characterized by a concave force-velocity relation.

We report on the characterization of actin driven lamellipodial protrusion forces and velocities in keratocytes. A vertically mounted glass fiber acted as a flexible barrier positioned in front of migrating keratocytes with parallel phase contrast microscopy. A laser beam was coupled into the fiber and allowed detecting the position of the fiber by a segmented photodiode. Calibration of the fiber was carried out with the thermal oscillation method. Deflection and force signals were measured during lamellipodial protrusion. Velocity was constant during initial contact whereas loading force increased until finally the cell was stalled at higher forces. Stall forces were on the order of 2.9 ± 0.6 nN, which corresponds to a stall pressure of 2.7 ± 1.6 nN/µm(2). Assuming a density of actin filaments of 240 filaments per µm, we can estimate a stall force per actin filament of 1.7 ± 0.8 pN. To check for adaption of the cell against an external force, we let the cell push toward the glass fiber several times. On the timescale of the experiment (∼1 min), however, the cell did not adapt to previous loading events.