Tilman E. Schäffer

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.

Small cantilevers for force spectroscopy of single molecules

We have used a simple process to fabricate small rectangular cantilevers out of silicon nitride. They have lengths of 9–50 μm, widths of 3–5 μm, and thicknesses of 86 and 102 nm. We have added metallic reflector pads to some of the cantilever ends to maximize reflectivity while minimizing sensitivity to temperature changes. We have characterized small cantilevers through their thermal spectra and show that they can measure smaller forces than larger cantilevers with the same spring constant because they have lower coefficients of viscous damping. Finally, we show that small cantilevers can be used for experiments requiring large measurement bandwidths, and have used them to unfold single titin molecules over an order of magnitude faster than previously reported with conventional cantilevers.

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.

Studies of vibrating atomic force microscope cantilevers in liquid

An atomic force microscope (AFM) design providing a focused spot of order 7 μm in diameter was used to analyze the motion of vibrating cantilevers in liquid. Picking an operating frequency for tapping mode AFM operation in liquid is complex because there is typically a large number of sharp peaks in the response spectrum of cantilever slope amplitude versus drive frequency. The response spectrum was found to be a product of the cantilever’s broad thermal noise spectrum and an underlying fluid drive spectrum containing the sharp peaks. The geometrical shape of transverse cantilever motion was qualitatively independent of the fluid drive spectrum and could be approximately reproduced by a simple theoretical model. The measurements performed give new insights into the behavior of cantilevers during tapping mode AFM operation in liquid.

Finite optical spot size and position corrections in thermal spring constant calibration

One of the most popular methods for calibrating the spring constant of an atomic force microscope cantilever is the thermal noise method. The usual implementation of this method has been to position the focused optical spot on or near the end of the cantilever, acquire a force curve on a hard surface to characterize the optical lever sensitivity and to then measure the thermal motion of the cantilever. The equipartition theorem then allows the spring constant to be calculated. In this work, we measured the spring constant as a function of the spot along the length of the cantilever. The observed systematic variation in the spring constant as a function of this position ranged from 15% for a short 60 μm cantilever up to 50% for a 225 μm cantilever we examined. In addition, the thermally calibrated spring constants systematically disagreed with spring constants calibrated using the Sader and Cleveland methods: by 50% for the short 60 μm cantilever and by 25% for the longest, 225 μm cantilever. By using a model that accounts for the spot diameter and position on the cantilever, the thermally measured spring constants were brought into better than 10% agreement with the other methods.

Practical implementation of dynamic methods for measuring atomic force microscope cantilever spring constants

Measurement of atomic force microscope cantilever spring constants (k) is essential for many of the applications of this versatile instrument. Numerous techniques to measure k have been proposed. Among these, we found the thermal noise and Sader methods to be commonly applicable and relatively user-friendly, providing an in situ, non-destructive, fast measurement of k for a cantilever independent of its material or coating. Such advantages recommend these methods for widespread use. An impediment thereto is the significant complication involved in the initial implementation of the methods. Some details of the implementation are discussed in publications, while others are left unsaid. Here we present a complete, cohesive, and practically oriented discussion of the implementation of both the thermal noise and Sader methods of measuring cantilever spring constants. We review the relevant theory and discuss practical experimental means for determining the required quantities. We then present results that compare measurements of k by these two methods over nearly two orders of magnitude, and we discuss the likely origins of both statistical and systematic errors for both methods. In conclusion, we find that the two methods agree to within an average of 4% over the wide range of cantilevers measured. Given that the methods derive from distinct physics we find the agreement a compelling argument in favour of the accuracy of both, suggesting them as practical standards for the field.

Magnetic Force Microscopy of the Submicron Magnetic Assembly in a Magnetotactic Bacterium

A magnetic force microscope (MFM) was used to image topography and magnetic forces from a chain of submicron single magnetic domain particles produced by and contained in isolated magnetotactic bacteria. The noncontact magnetic force microscope data were used to determine a value for the magnetic moment of an individual bacterial cell, of order 10−13 emu, consistent with the average magnetic moment of bacteria from the same sample, obtained by superconducting quantum interference device magnetometry. The results represent the most sensitive quantification of a magnetic force microscope image to date.

Comparison of Scanning Ion Conductance Microscopy with Atomic Force Microscopy for Cell Imaging

We present the first direct comparison of scanning ion conductance microscopy (SICM) with atomic force microscopy (AFM) for cell imaging. By imaging the same fibroblast or myoblast cell with both technologies in series, we highlight their advantages and disadvantages with respect to cell imaging. The finite imaging force applied to the sample in AFM imaging results in a coupling of mechanical sample properties into the measured sample topography. For soft samples such as cells this leads to artifacts in the measured topography and to elastic deformation, which we demonstrate by imaging whole fixed cells and cell extensions at high resolution. SICM imaging, on the other hand, has a noncontact character and can provide the true topography of soft samples at a comparable resolution.

In vitro differentiation of human dental follicle cells with dexamethasone and insulin

The dental follicle is an ectomesenchymally derived connective tissue harboring precursor cells for the tooth supporting apparatus. In this study, we examined gene expression of freshly isolated human dental follicle cells during osteogenic differentiation in vitro. These plastic adherent fibroblastic cells express Notch‐1, nestin and vimentin. We differentiated dental follicle cells with dexamethasone or insulin‐based protocols into membrane‐like structures containing mineralizing foci. An analysis of mineralized tissue with atomic force microscopy illustrated a bone and cementum‐like structure. A real‐time RT‐PCR analysis was developed to investigate expression of typical osteoblast or cementoblast related genes during differentiation. Gene expressions of osteocalcin (OCN), bone morphogenic protein (BMP)‐2 and nestin were increased during the both differentiation approaches. Our work demonstrates differentiation of dental follicle cells with an insulin‐based protocol for the first time.

Noncontact Measurement of the Local Mechanical Properties of Living Cells Using Pressure Applied via a Pipette

Mechanosensitivity in living biological tissue is a study area of increasing importance, but investigative tools are often inadequate. We have developed a noncontact nanoscale method to apply quantified positive and negative force at defined positions to the soft responsive surface of living cells. The method uses applied hydrostatic pressure (0.1-150 kPa) through a pipette, while the pipette-sample separation is kept constant above the cell surface using ion conductance based distance feedback. This prevents any surface contact, or contamination of the pipette, allowing repeated measurements. We show that we can probe the local mechanical properties of living cells using increasing pressure, and hence measure the nanomechanical properties of the cell membrane and the underlying cytoskeleton in a variety of cells (erythrocytes, epithelium, cardiomyocytes and neurons). Because the cell surface can first be imaged without pressure, it is possible to relate the mechanical properties to the local cell topography. This method is well suited to probe the nanomechanical properties and mechanosensitivity of living cells.