James B. Thompson

Molecular mechanistic origin of the toughness of natural adhesives, fibres and composites

Natural materials are renowned for their strength and toughness,,,,. Spider dragline silk has a breakage energy per unit weight two orders of magnitude greater than high tensile steel,, and is representative of many other strong natural fibres,,. The abalone shell, a composite of calcium carbonate plates sandwiched between organic material, is 3,000 times more fracture resistant than a single crystal of the pure mineral,. The organic component, comprising just a few per cent of the composite by weight, is thought to hold the key to nacres fracture toughness,. Ceramics laminated with organic material are more fracture resistant than non-laminated ceramics,, but synthetic materials made of interlocking ceramic tablets bound by a few weight per cent of ordinary adhesives do not have a toughness comparable to nacre. We believe that the key to nacres fracture resistance resides in the polymer adhesive, and here we reveal the properties of this adhesive by using the atomic force microscope to stretch the organic molecules exposed on the surface of freshly cleaved nacre. The adhesive fibres elongate in a stepwise manner as folded domains or loops are pulled open. The elongation events occur for forces of a few hundred piconewtons, which are smaller than the forces of over a nanonewton required to break the polymer backbone in the threads. We suggest that this ‘modular’ elongation mechanism might prove to be quite general for conveying toughness to natural fibres and adhesives, and we predict that it might be found also in dragline silk.

Bone indentation recovery time correlates with bond reforming time

Despite centuries of work, dating back to Galileo, the molecular basis of bones toughness and strength remains largely a mystery. A great deal is known about bone microsctructure and the microcracks that are precursors to its fracture, but little is known about the basic mechanism for dissipating the energy of an impact to keep the bone from fracturing. Bone is a nanocomposite of hydroxyapatite crystals and an organic matrix. Because rigid crystals such as the hydroxyapatite crystals cannot dissipate much energy, the organic matrix, which is mainly collagen, must be involved. A reduction in the number of collagen cross links has been associated with reduced bone strength and collagen is molecularly elongated (‘pulled’) when bovine tendon is strained. Using an atomic force microscope, a molecular mechanistic origin for the remarkable toughness of another biocomposite material, abalone nacre, has been found. Here we report that bone, like abalone nacre, contains polymers with ‘sacrificial bonds’ that both protect the polymer backbone and dissipate energy. The time needed for these sacrificial bonds to reform after pulling correlates with the time needed for bone to recover its toughness as measured by atomic force microscope indentation testing. We suggest that the sacrificial bonds found within or between collagen molecules may be partially responsible for the toughness of bone.

Probing protein-protein interactions in real time.

We have used a prototype small cantilever atomic force microscope to observe, in real time, the interactions between individual protein molecules. In particular, we have observed individual molecules of the chaperonin protein GroES binding to and then dissociating from individual GroEL proteins, which were immobilized on a mica support. This work suggests that the small cantilever atomic force microscope is a useful tool for studying protein dynamics at the single molecule level.

Fast imaging and fast force spectroscopy of single biopolymers with a new atomic force microscope designed for small cantilevers

Small cantilevers allow for faster imaging and faster force spectroscopy of single biopolymers than previously possible because they have higher resonant frequencies and lower coefficients of viscous damping. We have used a new prototype atomic force microscope with small cantilevers to produce stable tapping-mode images (1 μm×1 μm) in liquid of DNA adsorbed onto mica in as little as 1.7 s per image. We have also used these cantilevers to observe the forced unfolding of individual titin molecules on a time scale an order of magnitude faster than previously reported. These experiments demonstrate that a new generation of atomic force microscopes using small cantilevers will enable us to study biological processes with greater time resolution. Furthermore, these instruments allow us to narrow the gap in time between results from force spectroscopy experiments and molecular dynamics calculations.

Direct Observation of the Transition from Calcite to Aragonite Growth as Induced by Abalone Shell Proteins

The mixture of EDTA-soluble proteins found in abalone nacre are known to cause the nucleation and growth of aragonite on calcite seed crystals in supersaturated solutions of calcium carbonate. Past atomic force microscope studies of the interaction of these proteins with calcite crystals did not observe this transition because no information about the crystal polymorph on the surface was obtained. Here we have used the atomic force microscope to directly observe changes in the atomic lattice on a calcite seed crystal after the introduction of abalone shell proteins. The observed changes are consistent with a transition to (001) aragonite growth on a (1014) calcite surface.

Evidence that Collagen Fibrils in Tendons Are Inhomogeneously Structured in a Tubelike Manner

The standard model for the structure of collagen in tendon is an ascending hierarchy of bundling. Collagen triple helices bundle into microfibrils, microfibrils bundle into subfibrils, and subfibrils bundle into fibrils, the basic structural unit of tendon. This model, developed primarily on the basis of x-ray diffraction results, is necessarily vague about the cross-sectional organization of fibrils and has led to the widespread assumption of laterally homogeneous closepacking. This assumption is inconsistent with data presented here. Using atomic force microscopy and micromanipulation, we observe how collagen fibrils from tendons behave mechanically as tubes. We conclude that the collagen fibril is an inhomogeneous structure composed of a relatively hard shell and a softer, less dense core.

Atomic force microscopy study of living diatoms in ambient conditions

We present the first in vivo study of diatoms using atomic force microscopy (AFM). Three chain‐forming, benthic freshwater species –Eunotia sudetica, Navicula seminulum and a yet unidentified species – are directly imaged while growing on glass slides. Using the AFM, we imaged the topography of the diatom frustules at the nanometre range scale and we determined the thickness of the organic case enveloping the siliceous skeleton of the cell (10 nm). Imaging proved to be stable for several hours, thereby offering the possibility to study long‐term dynamic changes, such as biomineralization or cell movement, as they occur. We also focused on the natural adhesives produced by these unicellular organisms to adhere to other cells or the substratum. Most man‐made adhesives fail in wet conditions, owing to chemical modification of the adhesive or its substrate. Diatoms produce adhesives that are extremely strong and robust both in fresh‐ and in seawater environments. Our phase‐imaging and force‐pulling experiments reveal the characteristics of these natural adhesives that might be of use in designing man‐made analogues that function in wet environments. Engineering stable underwater adhesives currently poses a major technical challenge.

On the interpretation of force extension curves of single protein molecules

The atomic force microscope can be used to forcibly unfold and extend single polypeptide chains. The resulting force versus distance curves have been widely interpreted to arise from the loss of entropy that the unfolded polypeptide chain experiences as it is extended. Here, we have used Monte Carlo simulations of unfolded polypeptide chains to examine the average distance between the ends of a polypeptide chain as a function of the force that pulls these ends apart. We examine two types of experiments: (a) A rigid force-sensor (bead-type) experiment: The chain is subjected to a constant stretching force f and the resulting chain extension is measured. (b) A flexible force-sensor (cantilever-type) experiment: The force is measured by the deflection of a cantilever that is attached to one end of the chain. The total length of the chain plus the displacement of the cantilever is fixed. In case (b), in the limit of a large cantilever force constant, the entropic force f is related to the free energy of the c...

Atomic force microscope detector drift compensation by correlation of similar traces acquired at different setpoints

The atomic force microscope measures surface topography by maintaining a certain cantilever deflection or vibration amplitude as the cantilever is scanned over a sample surface. The desired cantilever deflection or amplitude is referred to as the setpoint, and is maintained by moving the sample toward or away from the cantilever. The signal from the cantilever deflection detector has a real component, due to cantilever deflection, and a drift component due to various sources of drift. We present a method of eliminating the drift component by sensing and correcting it in real time. Our method involves automatically changing the setpoint so as to maintain a certain set difference in the relative feature richness of two traces taken with slightly offset setpoints. We show how the system maintains a setpoint only 70 mV above minimum, perturb it with a gentle blow of air that causes 200 mV of detector drift, and observe its recovery within 13+−6 s.

Assessing the quality of scanning probe microscope designs

We present a method for assessing an atomic force microscopes (AFMs) ability to reject externally applied vibrations. This method is demonstrated on one commercial and two prototype AFMs. For optimally functioning AFMs, we find that the response to externally applied vibrations obeys a 1/ω2 frequency dependence. This 1/ω2 frequency dependence can be understood by modelling the mechanical system which connects the AFM cantilever and the sample under test as a simple harmonic oscillator. According to such a model, the resonant frequency of the mechanical system which connects the AFM cantilever and the sample under test determines an AFMs ability to reject externally applied, low-frequency vibrations.