B. Drake

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.

Using force modulation to image surface elasticities with the atomic force microscope

Using a new mode of scanning, the force modulation mode, surfaces are imaged by the atomic force microscope. The new contrast mechanism relies on variation in the surface elasticity. The cross section of a carbon fibre and epoxy composite is imaged, showing contrast between the two materials. Surface elasticity variations across the cross section of the fibre are revealed. A lateral modulation mode is used to highlight atomic steps in gold.

Atomic Force Microscopy of Liquid-Covered Surfaces: Atomic Resolution Images.

Images of graphite surfaces that are covered with oil reveal the hexagonal rings of carbon atoms. Images of a sodium chloride surface, protected from moisture by oil, exhibit a monoatomic step. Together, these images demonstrate the potential of atomic force microscopy (AFM) for studying both conducting and nonconducting surfaces, even surfaces covered with liquids. Our AFM uses a cross of double wires with an attached diamond stylus as a force sensor. The force constant is ≊40 N/m. The resonant frequency is ≊3 kHz. The lateral and vertical resolutions are 0.15 nm and 5 pm.

Motion and enzymatic degradation of DNA in the atomic force microscope

The dynamics and enzymatic degradation of single DNA molecules can now be observed with the atomic force microscope. A combination of two advances has made this possible. Tapping in fluid has reduced lateral forces, which permits the imaging of loosely adsorbed molecules; and the presence of nickel ions appears to form a relatively stable bridge between the negatively charged mica and the negatively charged DNA phosphate backbone. Continuous imaging shows DNA motion and the process of DNA degradation by the nuclease DNase I. It is possible to see DNase degradation of both loosely adsorbed and tightly adsorbed DNA molecules. This method gives images in aqueous buffer of bare, uncoated DNA molecules with lengths of only a few hundred base pairs, or approximately 100 nm in length.

From atoms to integrated circuit chips, blood cells, and bacteria with the atomic force microscope

The atomic force microscope (AFM) can now bridge the gap from imaging objects that can be seen with an optical microscope to imaging atoms: a range in magnification of 104. High magnification images of germanium show single atoms separated by 0.4 nm while low magnification images of entire cells and portions of an integrated circuit chip provide lateral and vertical information over a range of 25 μm.

Immobilized proteins in buffer imaged at molecular resolution by atomic force microscopy

Samples of supported planar lipid-protein membranes and actin filaments on mica were imaged by atomic force microscopy (AFM). The samples were fully submerged in buffer at room temperature during imaging. Individual proteins bound to the reconstituted membrane were distinguishable; some structural details could be resolved. Also, surface-induced, self-assembling of actin filaments on mica could be observed. Monomeric subunits were imaged on individual actin filaments. The filaments could be manipulated on or removed from the surface by the tip of the AFM. The process of the decoupling of the filamentous network from the surface upon changing the ionic conditions was imaged in real time.

Wet lipid-protein membranes imaged at submolecular resolution by atomic force microscopy

Abstract We have employed an AFM to determine the structural properties of supported planar membranes and membrane-bound proteins in an aqueous environment. Images of an asymmetric Langmuir Blodgett film of a charged phospholipid show long range positional as well as orientational order; individual headgroups are resolved. In order to study biofunctional membranes we have employed a recently introduced technique that allows the controlled formation of planar lipid-protein membranes on solid supports from a vesicle suspension. Combining this technique with the AFM permits the nondestructive imaging of these models of cell membranes at molecular resolution under physiological conditions of ionic strength and temperature.

The bone diagnostic instrument II: Indentation distance increase

The bone diagnostic instrument (BDI) is being developed with the long-term goal of providing a way for researchers and clinicians to measure bone material properties of human bone in vivo. Such measurements could contribute to the overall assessment of bone fragility in the future. Here, we describe an improved BDI, the Osteoprobe IItrade mark. In the Osteoprobe IItrade mark, the probe assembly, which is designed to penetrate soft tissue, consists of a reference probe (a 22 gauge hypodermic needle) and a test probe (a small diameter, sharpened rod) which slides through the inside of the reference probe. The probe assembly is inserted through the skin to rest on the bone. The distance that the test probe is indented into the bone can be measured relative to the position of the reference probe. At this stage of development, the indentation distance increase (IDI) with repeated cycling to a fixed force appears to best distinguish bone that is more easily fractured from bone that is less easily fractured. Specifically, in three model systems, in which previous mechanical testing and/or tests reported here found degraded mechanical properties such as toughness and postyield strain, the BDI found increased IDI. However, it must be emphasized that, at this time, neither the IDI nor any other mechanical measurement by any technique has been shown clinically to correlate with fracture risk. Further, we do not yet understand the mechanism responsible for determining IDI beyond noting that it is a measure of the continuing damage that results from repeated loading. As such, it is more a measure of plasticity than elasticity in the bone.

Tunneling microscope for operation in air or fluids

A tunneling microscope that is a hybrid between IBM Zurich designs and squeezable tunnel junctions has been operated in air, oil, and liquid nitrogen. Key design goals were (1) maximum rigidity and (2) minimum thermal drift. Images of individual atoms in a close packed layer have been obtained under liquid nitrogen.

Scanning ion conductance microscope

In 1981, the age of the scanning probe microscopes (SPMs) began when Binnig, Rohrer, and cowokers developed the first scanning tunneling microscope (STM) [1]. Their setup was based on measuring an electrical tunneling current between a sharp metal tip and a conducting sample. For the first time, a sample surface could be imaged with true atomic resolution in real space. The STM launched the development of several other types of SPMs. In general, these microscopes consist of a small, submicrometer probe, which senses a certain physical interaction with the sample and which is scanned over the sample to generate an image. For example, Pohl et al. invented the scanning near-field optical microscope (SNOM) in 1984 [2], which uses an evanescent electromagnetic field in the subwavelength range to image the sample. In 1986, Binnig and co-workers developed the atomic force microscope (AFM), which is based on measuring the mechanical forces between a sharp tip and the sample [3]. The AFM is not limited to conducting or transparent samples and has become one of the most important tools in nanoscale science. The AFM also works in aqueous environments, such as buffer solutions and so is well suited for biological samples [4]. Since then, several related SPMs have been developed, such as the magnetic force microscope [5,6] the electrical force microscope [7], and the scanning electrochemical force microscope (SECM) [8].