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.

To introduce this special issue of the Journal of Molecular Recognition dedicated to the applications of atomic force microscopy (AFM) in life sciences, this paper presents a short summary of the history of AFM in biology. Based on contributions from the first international conference of AFM in biological sciences and medicine (AFM BioMed Barcelona, 19-21 April 2007), we present and discuss recent progress made using AFM for studying cells and cellular interactions, probing single molecules, imaging biosurfaces at high resolution and investigating model membranes and their interactions. Future prospects in these different fields are also highlighted.

A flash‐induced transthylakoid electric field was measured at 515 nm as an electrochromic absorbance shift in intact potato leaves using a double flash differential spectrophotometer. The decay rate of the electrochromic shift in dark‐adapted samples was used to examine the conductance to ions of thylakoid membranes. Heat stress (39.5 °C for 15 min) was found to accelerate drastically the electric field decay, with the half decay time falling from more than 200 ms to less than 45 ms. Heat‐induced acceleration of the electric field breakdown was insensitive to the PSII electron donor Hydroxylamine and to the ATPase inhibitor dicyclohexylcarbodiimide (DCCD), thus indicating that it reflects an increase in thylakoid membrane permeability after heat stress. This phenomenon did not involve peroxidative damage of membrane lipids. Acceleration of the electric field relaxation exhibited the same temperature dependence as that of PSII deactivation, suggesting that the ionic permeability of thylakoid membranes is one of the most heat‐sensitive components of the photosynthetic apparatus. When potato leaves were infiltrated with 100 mol m−3 ascorbate (in a buffer of pH 5), there was massive conversion of the carotenoid violaxanthin to zeaxanthin. This change in carotenoid composition protected thylakoid membranes against heat‐induced changes in permeability, as revealed by the maintenance of a slow decay of the 515 nm absorbance change after heat stress. No such effect was observed after treatments which did not induce the vio‐laxanthin‐to‐zeaxanthin conversion: leaf infiltration with 0 mol m−3 ascorbate (at pH 5 or 8), 100 mol m−3 ascorbate at pH 8 or 100 mol m−3 ascorbate +5 mol m−3 dithiothreitol at pH 5. Increased stability of the permeability properties of thylakoid membranes was also observed after a mild heat treatment (2 h at 35 °C). The data presented suggest that de‐epoxidized xanthophylls in vivo stabilize thylakoid membranes and protect thylakoids against heat‐induced disorganization.

Thanks to Dynamic Force Spectroscopy (DFS) and developments of massive data analysis tools, such as YieldFinder, Atomic Force Microscopy (AFM) becomes a powerful method for analyzing long lifetime ligand-receptor interactions. We have chosen the well-known system, (strept)avidin-biotin complex, as an experimental model due to the lack of consensus on interpretations of the rupture force spectrum (Walton et al., 2008). We present new measurements of force-displacement curves for the (strept)avidin-biotin complex. These data were analyzed using the YieldFinder software based on the Bell-Evans formalism. In addition, the Williams model was adopted to interpret the bonding state of the system. Our results indicate the presence of at least two energy barriers in two loading rate regimes. Combining with structural analysis, the energy barriers can be interpreted in a novel physico-chemical context as one inner barrier for H-bond ruptures ( <1 Å), and one outer barrier for escaping from the binding pocket which is blocked by the side chain of a symmetry-related Trp120 in the streptavidin tetramer. In each loading rate regime, the presence of multiple parallel bonds was implied by the Williams model. Interestingly, we found that in literature different terms created for addressing the apparent discrepancies in the results of avidin-biotin interactions can be reconciled by taking into account multiple parallel bonds.

We have found that the only high redox potential electron transfer component in the soluble fraction of Rubrivivax gelatinosus TG-9 is a high-potential iron-sulfur protein (HiPIP). We demonstrated the participation of this HiPIP in the photoinduced electron transfer both in vivo and in vitro. First, the addition of HiPIP to purified membranes enhanced the rate of re-reduction of the photooxidized reaction center. Second, the photooxidation of HiPIP was observed in intact cells of Ru. gelatinosus TG-9 under anaerobic conditions by EPR and absorption spectroscopies. Analysis of flash-induced absorption changes showed that the equilibration of positive equivalents between the reaction center and HiPIP occurs in less than 1 ms after flash excitation. The complete re-reduction of the photooxidized reaction center is achieved in tens of milliseconds. The turnover of a cyt bc1 is also involved in this reaction, as shown by a slow electrogenic phase of the membrane potential linked to this process.

We used dynamic force spectroscopy (DFS) to explore the energy landscape of interactions between a chelated uranyl compound and a monoclonal antibody raised against the uranyl-dicarboxy-phenanthroline complex. We estimated the potential energy barrier widths and the relevant thermodynamic rate constants along the dissociation coordinate. Using atomic force microscopy, four different experimental setups with or without the uranyl ion in the chelate ligand, we have distinguished specific and nonspecific binding in the binding affinity of the uranyl compound to the antibody. The force loading rates for our system were measured from 15 to 26,400 pN/s. The results showed two regimes in the plot of the most probable unbinding force versus the logarithm of the loading rate, revealing the presence of two (at least) activation barriers. Analyses of DFS suggest parallel multivalent binding present in either regime. We have also built a molecular model for the variable fragment of the antibody and used computational graphics to dock the chelated uranyl ion into the binding pocket. The structural analysis led us to hypothesize that the two regimes originate from two interaction modes: the first one corresponds to an energy barrier with a very narrow width of 0.5 +/- 0.2 A, inferring dissociation of the uranyl ion from its first coordination shell (Asp residue); the second one with a broader energy barrier width (3.9 +/- 0.3 A) infers the entire chelate compound dissociated from the antibody. Our study highlights the sensitivity of DFS experiments to dissect protein-metal compound interactions.

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Summary Classical structural biology techniques face a great challenge to determine the structure at the atomic level of large and flexible macromolecules. We present a novel methodology that combines high-resolution AFM topographic images with atomic coordinates of proteins to assemble very large macromolecules or particles. Our method uses a two-step protocol: atomic coordinates of individual domains are docked beneath the molecular surface of the large macromolecule, and then each domain is assembled using a combinatorial search. The protocol was validated on three test cases: a simulated system of antibody structures; and two experimentally-based test cases: Tobacco mosaic virus, a rod-shaped virus; and aquaporin Z, a bacterial membrane protein. We have shown that AFM-intermediate resolution topography and partial surface data are useful constraints for building macromolecular assemblies. The protocol is applicable to multi-component structures connected in the polypeptide chain or as disjoint molecules. The approach effectively increases the resolution of AFM beyond topographical information down to atomic-detail structures.